Alexa fluor 594 affinipure donkey anti mouse igg h l

The paraffin-embedded tissue specimens were sectioned into 5 μm thick sections. After deparaffinization, rehydration through graded ethanol, and antigen retrieval with pH 6.0 citrate buffer in a pressure cooker for 15 mins, sections were blocked with 5% bovine serum albumin (BSA) for 1 hour at room temperature and then incubated with diluted primary antibodies in 1% BSA at 4 °C overnight. Anti-smooth muscle 22α (ab14106, ABCAM, 1:50) and anti-smooth muscle heavy chain (ab53219, ABCAM, 1:50) were used for detecting smooth muscle contractile proteins, anti-von Willebrand factor (anti-vWF (A0082, DAKO, 1:200) and anti-CD31 (NB100–2284, Novus Biologicals, 1:100) for endothelial cell markers, anti-CD68 (ab31630, ABCAM, 1:100) for macrophage cell marker and anti- IL-1 beta (NB600–633, Novus Biologicals, 1:100) for pro-inflammatory cytokine. Alexa Fluor® 647 AffiniPure Donkey Anti-Mouse IgG (H+L) (715–605-151, Jackson Immuno Research, 1:500), Alexa Fluor® 647 AffiniPure Donkey Anti-Rabbit IgG (H+L) (711–605-152, Jackson Immuno Research, 1:500), Alexa Fluor® 594 AffiniPure Donkey Anti-Mouse IgG (H+L) (715–585-150, Jackson Immuno Research, 1:500) were used as secondary antibodies. The sections were counterstained with DAPI before being observed under an immunofluorescence microscope (DP80 microscope digital camera, Olympus).

Mice were anesthetized with an i.p. injection of 240 mg/kg Avertin and transcardially perfused with ice-cold 4% paraformaldehyde in phosphate buffer (pH 7.4, 150 ml per mouse). Brains were removed and post-fixed for 24 h in the same fixative and then immersed for 24 h each in 20 and 30% sucrose in phosphate buffer. Brains were frozen and 50 μm sections were cut for use in free-floating immunohistochemistry (Jovasevic et al., 2015 (link)) with primary antibodies against mCherry (1:1000, rabbit, Abcam, AB167453), Tyrosine hydroxylase (1:2000, Immunostar, 22941), Calretinin (1:4,000, Swant, CG1), GABA_AR-δ (1:2000, Alomone, AGA-014). Secondary antibodies were obtained from Jackson ImmunoResearch (1:200, Alexa Fluor® 594 AffiniPure Donkey Anti-Mouse IgG [H+L]). GFP was visualized by its intrinsic fluorescence. Nuclei were counterstained with Hoechst 33342 (ThermoFisher), except in double staining experiments, where blue color was used for one of the antibodies. Sections were mounted using FluorSave (Millipore-Sigma) and observed with Leica microscope equipped with a CCD (Olympus) camera, using 5 ×, 10 ×, or 20 × objectives. For light microscopy, signals were visualized with diaminobenzidine (Sigma).

Free-floating coronal sections (40 μm) from rats with viral injection of pAAV_2/5-Syn1-ChRERα into the right PrL/ACd or left M1 and free-floating horizontal sections (80 μm) from squirrel monkey with viral injection of pAAV2/5-hSyn-ChRERα into the left M1 were incubated for 1 hour in phosphate buffer (PB) supplemented with 4% BSA and 0.3% Triton X-100. Sections were then incubated with the specific primary antibody, mouse anti-ChR2 (1:250; American Research Products Inc., 03-651180), overnight at 4°C. After rinsing 3× for 10 min in PB, sections were incubated in the fluorescence secondary antibody Alexa Fluor 594 AffiniPure donkey anti-mouse IgG (H+L) (1:100; Jackson ImmunoResearch Laboratories Inc., 715-585-151) for 2 hours at RT. After rinsing, sections were mounted with mounting medium with DAPI on slides. Fluorescent images were collected with the Zeiss LSM880 with Cy7.5 Confocal System (Zeiss). Images were taken sequentially with different lasers with a 20× objective. This experiment was successfully repeated three times.